Synthesis, Characterization And Biological Evaluation Of Biomimetic Phosphorylcholine-Bound Chitosan Derivatives

Through constructing a cell outer membrane mimetic structure, a novel phosphorylatedchitosan derivatives were designed to couple phosphorylcholine(PC) with bioactivity ontochitosan with a phosphamide binding, which can be used in biomedical materials.Three phosphorylated methods of chitosan were investigated, including (A)1,1,1,3,3,3-Hexafluoro-2-propanol used as reaction medium to synthesize phosphorylatedchitosan directly based on Antherton-Todd reaction,(B) using Cs-NPTh as the intermediate andphosphorylcholine dichloride as phosphorylated agent,(C) Cs-Tr as the intermediate tosynthesize phosphorylated chitosan based on Antherton-Todd reaction. It was found thatphosphorylated chitosan can be synthesized using method (C).Using Antherton-Todd reaction, phosphorylated chitosan derivatives with different DSwere synthesized. The new peaks in NMR and FTIR spectra indicated that the PC moiety hadbeen conjugated to the amino group of the chitosan. The DS of PC moiety was calculated by theamount ratio of H of-N+(CH3)3(from PC) to H1(from glucosamine units of GlcN andGlcN-PC) based on the1H NMR spectra. The DS values ranged from16to42mol%. The GPCanalysis found that PCCs appeared lower Mw values but higher Mw/Mn values compared withthe starting chitosan. All these PCCs with decreased crystallization showed excellent solubilityin the aqueous solutions within a wide pH range (1-12). TGA and DSC results revealed that thethermal stability of PCCs decresed with the increase of DS value.The in vitro cycotoxicity of PCCs copolymers was evaluated using a MTT assayperformed with NIH/3T3cells. The cells showed almost100%viability in the presence ofPCCs with different DS value, which suggests that all the PCCs with low toxicity are safety forbiomedical application. The blood compatibility were evaluated by means of blood-clotting andplatelet adhesion assay. The blood-clotting assay indicated that PCCs could prolong theblood-clotting process. Platelet adhesion assay showed that PCCs could effectively inhibit theplatelet adhesion and activation. Using bovine serum albumin (BSA) as a model protein, UVadsorption spectra and fluorescence spectra revealed that the non-specific interactions betweenPCCs and BSA were effectively suppressed and the conformation of BSA was almostunchanged with the addition of PCCs. Further, PCCs nanoparticles could be still formed in a spherical shape similar to chitosannanoparticles with zeta potential between18-28mV by ionically crosslinking withtripolyphosphate (TPP), the sizes were in the ranges of60-120nm. In addition, the amphiphilicPCCs copolymers could self-assemble to form spherical nano-aggregates. The CMC values ofPCCs were in the range of0.129-0.256mg/mL. The sizes of PCCs self-aggregates with zetapotential between0-4mV were in the ranges of30-60nm. These PCCs nanoparticles could beused as promising delivery systems for drug or gene delivery applications.